Expansion Microscopy for Cell Biology Analysis in Fungi

Abstract

Super-resolution microscopy has evolved as a powerful method for subdiffractionresolution fluorescence imaging of cells and cellular organelles, but requires sophisticated and expensive installations. Expansion microscopy (ExM), which is based on the physical expansion of the cellular structure of interest, provides a cheap alternative to bypass the diffraction limit and enable super-resolution imaging on a conventional fluorescence microscope. While ExM has shown impressive results for the magnified visualization of proteins and RNAs in cells and tissues, it has not yet been applied in fungi, mainly due to their complex cell wall. Here we developed a method that enables reliable isotropic expansion of ascomycetes and basidiomycetes upon treatment with cell wall degrading enzymes. Confocal laser scanning microscopy (CLSM) and structured illumination microscopy (SIM) images of 4.5-fold expanded sporidia of Ustilago maydis expressing fluorescent fungal rhodopsins and hyphae of Fusarium oxysporum or Aspergillus fumigatus expressing either histone H1-mCherry together with Lifeact-sGFP or mRFP targeted to mitochondria, revealed details of subcellular structures with an estimated spatial resolution of around 30 nm. ExM is thus well suited for cell biology studies in fungi on conventional fluorescence microscopes.

Single-Molecule FISH Reveals Subcellular Localization of α-Amylase and Actin mRNAs in the Filamentous Fungus Aspergillus oryzae

“…Recently, expansion microscopy (ExM) has been successfully applied to fungal cells to visualize details of subcellular structures at a resolution of around 30 nm ( Götz et al, 2020 ). Although similar to smFISH, ExM is also limited to available fluorophores and to fixed cells ( Tillberg et al, 2016 ), physical expansion allows the sample to be observed at super-resolutions with conventional microscopy settings.…”

Current and future advances in fluorescence-based visualization of plant cell wall components and cell wall biosynthetic machineries

“…Application of this method to organisms with strong cell walls lags significantly behind its use with softer cell types, but there are a few reported examples. Several fungal species were recently successfully expanded [ 56 ], and the technique was also used in plants to image the chromatin ultrastructure in barley [ 57 ], and aspects of transcription regulation during Arabidopsis embryo fertilization [ 58 ]. With continued method development, this technique has a strong potential to enable nanoscale fluorescent imaging of otherwise inaccessible cell wall epitopes.…”

Prospects and limitations of expansion microscopy in chromatin ultrastructure determination

“…Since then, several expansion protocols emerged to increase the expansion factor and to preserve the ultrastructural features. These protocols were adapted to species like fungi, human, mouse, fruit fly and zebrafish and soft tissues such as brain, skin, kidney and liver (Chen et al 2015 ; Tillberg et al, 2016 ; Cahoon et al 2017 ; Freifeld et al 2017 ; Halpern et al 2017 ; Jiang et al 2018 , Lim et al 2019 ; Truckenbrodt et al, 2019 ; Götz et al 2020 ; Zwettler et al 2020b ). The following processes occur during ExM to fix, embed and expand the specimen successfully: (1) during the fixation with a formaldehyde/acrylamide mixture, formaldehyde crosslinks proteins/DNA/RNA to each other; (2) during gelation, the crosslinked proteins become crosslinked to the polyacrylamide (PAA) gel due to the acrylamide provided during fixation; (3) during denaturation in SDS buffer and at high temperature, all crosslinked proteins denature while remaining crosslinked to the PAA gel mesh which starts to expand in the denaturation buffer; (4) during expansion in water, all proteins renature back with gaps between each other but still bound to the PAA gel mesh preserving their exact position as before expansion (Chen et al 2015 ; Cho et al 2018 ; Tillberg and Chen 2019 ; Wassie et al 2019 ).…”